Bulletin UASVM, Veterinary Medicine 65(2)/2008 pissn 1843-5270; eissn 1843-5378 BONE MARROW CELLS VERSUS MATURE LYMPHOCYTES AND MONOCYTES CAPACITY OF IMMUNOTOLERANCE INDUCTION IN COBB 500 HYBRIDS Şereş Monica 1, C. Igna 1, D. Cioca 2, Larisa Schuszler 1 1 University of Agricultural Sciences and Veterinary Medicine of Banat, 119 Calea Aradului, 300645- Timisoara, Romania 2 University of Medicine and Pharmacy Victor Babeş Timisoara, Centre of Immunophysiology and Biotechnology seres_monica@yahoo.com Keywords: bone marrow cells, lymphocytes, monocytes, immunotolerance, skin grafts Abstract. The purpose of this study was the comparison between capacity of bone marrow cells on the one hand, and of mature lymphocytes and monocytes on the other hand, to induce immunotolerance by inoculation in COBB 500 hybrids at different embryonic stages. Antigenic material was obtained by aspiration of tibial and femoral bone marrow, respectively by blood sampling from the donor birds (Ross hybrids).we used 240 embryonated eggs: 60 eggs for in ovo inoculation with bone marrow cells suspension in the 5 th day of embryo development and 60 for inoculation in the same way and day with mature lymphocytes and monocytes suspension; the rest of biologic material was inoculated with these suspensions in allantoidal vessel in the 8 th day of the embryo. Seven days after the full-thickness skin grafts transplantation (from the donor birds) the lymphocyte T subsets of the recipient birds were determined by flow cytometry and immunomagnetic sorting. Also, all the skin grafts were periodically monitored for macroscopic characteristics. This experiment demonstrates the superiority of bone marrow cells in immunotolerance induction in birds after inoculation in 5 th day of embryonic development. INTRODUCTION This experimental study represents the first report about the comparison between capacity of bone marrow cells and mature lymphocytes and monocytes to induce immunotolerance in recipient bird and about the inoculation method for triggering this phenomenon. MATERIAL AND METHODS The biologic material was represented by six one year old Ross hybrids (donor birds for antigenic material and for skin grafts) by 240 embryonated eggs (COBB 500 hybrids). The raw antigenic material was obtained by aspiration of tibial and femoral bone marrow, respectively by blood sampling from the donor birds. The antigenic material, suspension of bone marrow cells and mature lymphocytes and monocytes, was obtained by three centrifugations of biological samples after Ficoll-Paque solution addition and PBS dilution.distribution of biologic material regarding to the moment of inoculation and the method used: 60 eggs for in ovo inoculation (fig. 1) with bone marrow cells suspension in the 5 th day of embryonic development and 60 for inoculation in the same way and day with mature lymphocytes and monocytes suspension; the rest of biologic material was inoculated - 60 eggs with bone marrow cells suspension and 60 eggs with mature lymphocytes and monocytes suspension - in allantoidal vessel (fig. 2) of 8 th days embryos. 222
Fig.1. In ovo inoculation Fig.2. Inoculation in allantoidal vessel at 8th days embryo Transplantation of the full-thickness allogenic skin graft was made at the age of four weeks, respecting the principles suggested by Swaim, 1993 and Shannon, 2002. The subjects were examined periodically, paying attention to the 21 macroscopic characteristics of the skin grafts, making different measurements and taking pictures. One week after transplant, form all individuals were gathered peripheral blood for obtaining the T lymphocytes using the Ficoll-Paque solution for separation. T lymphocytes were labeled with monoclonal antibodies as follows: antibodies anti-cd3 for label T lymphocytes; antibodies anti-cd4 for label T helper lymphocytes; antibodies anti-cd8 for label cytotoxic T cells; antibodies anti-cd44ra for differentiating between memory and naive T cells; antibodies anti-cd28 for differentiating between memory and effector T lymphocytes; and antibodies anti-cd25 for label the eventual activate subset of T cells from subpopulation Treg regulatory T cells. This labeling served in determining the lymphocytes T profile in all groups of recipient birds. By flow cytometry, the cells population labeled in four colors (CD3FITC, CD4PE CD45RAPerCP, and CD28APC for determining the T helper subsets and CD3FITC, CD8PE, CD45RAPerCP, and CD28APC for determining the T cytotoxic subsets) was quantitative analyzed in all groups. The lymphocytes subsets were defined as follows: naive CD4+ helper T cells with phenotype CD3+CD4+CD45RA+CD28+; memory CD4+ helper T cells with phenotype CD3+CD4+CD45RA-CD28+; effector CD4+ helper T cells with phenotype CD3+CD4+CD45RA-CD28-; effector CD4+ helper T cells with phenotype CD3+CD4+ CD45RA+CD28-; naive CD8+ cytotoxic T cells with phenotype CD3+CD8+CD45RA+CD28+; memory CD8+ cytotoxic T cells with phenotype CD3+CD8+CD45RA-CD28+; effector CD8+ cytotoxic T cells with phenotype CD3+CD8+CD45RA-CD28-; and effector CD8+ cytotoxic T cells with phenotype CD3+CD8+CD45RA+CD28-. Immunomagnetic sorting of the lymphocyte T subsets for establishing the Treg subpopulation was made with Dynal immunomagnetic beads coupled with CD3, CD4, and CD8, and after that the subsets were labeled with CD45RA or CD45RO (according to the case), and also with CD28, CD5, and CD25. RESULTS AND DISCUSSIONS We appeal to the inoculation of antigenic material in the 5th and 8th days of embrionary development, knowing that the hematopoietic stem cells from the vitellus membrane migrates in the thymus and the Fabricius bursa, under the influence of some chemo-tactic factors, on days 5-7 of incubation (2, 8, 9) and also knowing that a tardy inoculation is unhelpful for immunotolerance induction (6). These cells get differentiated and develop in bursal follicles until the 12th day of incubation, so that in days 14-16 appear the first cells that present IgM on the surface of the membrane, capable of tying antigens (2, 8, 9). The lymphocytes that 223
present membrane IgG develop a little later, around the hatching time (8, 9). Thus, it can be considered that the embryo is immunocompetent starting with the 14th day of the embrionary development (2, 8, 9). The transplant has been executed at the age of four weeks due to the fact that an earlier intervention, especially during the first week of life, could have led to the acceptance of an incompatible allogenic graft, even in the case of the absence of the induction of the immunotolerance (1). The usage of a donor bird from a different line (Ross hybrid) than the one of the recipients (COBB 500 hybrids) excluded the existence of all the factors that could have induced the complete acceptance or a late rejection, as the total or partial MHC compatibility (3). Results of inoculation with mature lymphocytes and monocytes suspension The 5 th day in ovo inoculation caused the death of all embryos in the interval between 8 and 10 days of incubation. The mechanisms involved must have been a graft versus host (spleen enlargement) one or a traumatic one (injection) because 20% of dead embryo had celosomy. After the inoculation in allantoidal vessel of 8 days embryos we obtained only six viable poultry. The death of the rest of embryo intervened between 14 and 18 days of incubation and lesions (hemorrhagic lesions in liver, spleen and gut, spleen enlargement) suggest the implication of graft versus host mechanisms (9). All the skin grafts transplanted on these poultry were rejected between 7 and 10 days after transplant. In figure 3 is presented one example of macroscopic characteristic evolution in ten days. Fig.3. Macroscopic characteristic of skin graft evolution in ten days: a first day after transplant; b second day; c third day; d fourth day; e seventh day; f tenth day The rejection of the full-thickness skin allografts in the group which was inoculated in 8 th days of embryo development took place in a time interval comparable with the existent data in the speciality literature about allogenic grafts: up to seven days in the case of the total incompatibility of B complex, respectively up to 15 days in the case of the incompatibility only of the complex Rfp-Y (7) or, in opinion of other authors, in 10-14 days (6, 8, 9). Flow cytometry didn t show a significant decrease in naive T cells subset (CD3 + CD4 + CD45RA + CD28 + for helper T cells and CD3 + CD8 + CD45RA + CD28 + for cytotoxic T cells) and an increase of the memory T lymphocytes (CD3 + CD4 + CD45RA - CD28 + for helper T cells and CD3 + CD8 + CD45RA - CD28 + for cytotoxic T cells), as well as not showing an increase in effector T cells (CD3 + CD4 + CD45RA - CD28 - and CD3 + CD4 + CD45RA + CD28 - for helper T lymphocyte or CD3 + CD8 + CD45RA - CD28 - and CD3 + CD8 + CD45RA + CD28 - for cytotoxic T cells) - fig. 4. 224
Fig. 4. Examples of T cells subsets distribution in poultry inoculated in 8 th day of embryonic development with mature lymphocytes and monocytes suspension Results of inoculation with bone marrow cells suspension After the in ovo inoculation in the 5 th day were obtained 13 viable poultry. The death of the rest of embryo intervened in the interval between 7 (with celosomy, suggesting a traumatic mechanism) and 18 days (with no apparent lesions) of incubation. All the skin grafts transplanted on these poultry were rejected between 15-20 (31%) and 25-27 days (69%) after transplant. In figure 5 is presented one example of macroscopic characteristic evolution in 27 days. Fig.5. Macroscopic characteristic of skin graft evolution in 27 days: a first day after transplant; b third day; c fifth day; d seventh day; e 11 th day; f 20 th day; g 27 th day Flow cytometry showed a significant decrease in naive T cells subset, together with a significant increase of the memory T lymphocytes, as well as a significant increase in effector T cells (fig. 6). Fig. 6. Examples of T cells subsets distribution in poultry inoculated in 5 th day of embryonic development with bone marrow cells suspension 225
Immunomagnetic sorting permitted a more detailed characterization of the CD25 + subsets existing in peripheral blood (fig. 7). Almost all CD25 + cells belong to T helper subpopulation (CD4 + ) suggesting that are T regulatory lymphocytes implicated in immunotolerance induction in embrionary life beside the antigens. Also, the great majority of the CD25 + T cells (98%) are CD28 + suggesting that these belong to the memory subsets and not to the effector subsets. Fig.7. Results of immunomagnetic sorting of T cells subsets After the inoculation in allantoidal vessel in 8 th day embryos we obtained only 11 viable poultry. The death of the rest of embryo intervened in the interval between 12 and 19 days of incubation without apparent lesions. The full-thickness skin grafts evolution and the results of flow cytometry didn t show major differences between this group and the poultry inoculated with mature lymphocytes and monocytes in the same day all the grafts were rejected until 12 days and the comparison of the flow cytometry results demonstrate the similarity in T cells subsets pattern. CONCLUSIONS Mature lymphocytes and monocytes didn t prove themselves to be capable to induce the immunotolerance independently on tardily or early inoculation method used. In addition, the embryos lesions demonstrated that these mature cells generate the graft versus host phenomena. Using bone marrow cells for inoculation in 5 th day of embryos development delayed the skin grafts reject until 27 days; this overrunning by far the data presents in speciality literature about allograft rejection. This aspect, together with the flow cytometry and immunomagnetic sorting results prove the immunotolerance induction. Because the reject was only delayed and not annulated, is required an earlier inoculation or a replacement technique which allows intervention in earlier embrionary stages. The fast reject in poultry inoculated with bone marrow cells in the 8 th day of embrionary development and the minor differences between this group and the one inoculated with 226
mature lymphocytes and monocytes in the same day proves that the injection was made when immune system of the embryos was partially operational. BIBLIOGRAPHY 1. Billingham R.E., H.K. Poole, W.K. Silvers, 1961, Transplantation Immunity, Immunological Tolerance, and Chicken x Turkey Interspecific Hybrids, Proceedings of the National Academy of Sciences of the United States of America, 47, 7, 1039-1043. 2. Jankovic B.D., Katarina Isakovic, M.L. Lukic, N.L. Vujanovic, Spomenka Petrovic, B. M. Markovic, 1975, Immunological capacity of the chicken embryo, Immunology, 29, 497-508. 3. Lehtonen L., O. Vainio, T. Veromaa, P. Toivanen, 1989, Tolerance to class I major histocompatibility complex antigens in chicken B cell chimeras. Effect of B cell depletion on transferability of tolerance, European Journal of Immunology, 19, 3, 425-432. 4. Shannon T.F., 2002, Avian integumentary surgery, Seminar in Avian and Exotic Pet Medicine, 11(3):125-135. 5. Swaim S.F., 1993, Skin grafts, in: Text Book of Small Animal Surgery, ed. Slatter D., vol. I, 2nd edition, W.B. Saunders Company, Philadelphia. 6. Şereş Monica, A. Sala, Larisa Schuszler, H. Sărăndan, Roxana Dascalu, M. Sabău, Simona Anghel, I. Checiu, D. Cioca, C. Igna, 2007, Evolution of full-thickness and pinch skin allografts evaluation at Cobb 500 hybrids after immunotolerance induction in the ninth day of embryonary development, Bul. USAMV Cluj-Napoca, vol. 64(1-2), 539-544. 7. Thraval P., M. Afanassieff, D. Bouret, G. Luneau, E. Esnault, 2003, Role of nonclassical class I genes of the chicken major histocompatibility complex Rfp-Y locus in transplantation immunity, Immunogenetics, 55, 9, 647-651. 8. Tizard I., 1992, Veterinary Immunology. An Introduction, 4 th Edition, W.B. Saunders Company, Philadelphia. 9. Tizard I, 2008, Veterinary Immunology. An Introduction, 8 th Edition, W.B. Saunders Company, Philadelphia. 227